Project Summary/Abstract Mucosal associated invariant T (MAIT) cells are an innate-like T cell subset prevalent in humans and enriched in the airway. Human MAIT cells have been defined by the expression of the semi-invariant TCR? chain TRAV1- 2/TRAJ12/20/33 and their restriction by the non-polymorphic MHC class I-like molecule, MHC-related protein 1 (MR1). MAIT cells recognize Mtb and can be activated by small organic molecules, derived from the riboflavin biosynthesis pathway. We have shown that MR1-restricted T cells can use TCRs that are not TRAV1-2, and can recognize organisms (S. pyogenes) that cannot produce riboflavin. Consequently, we define MAIT cells as a subset of MR1-restricted T cells (MR1Ts). Furthermore, we find that not all MR1Ts can be defined based on MR1 tetramer bound to the known MAIT agonist / MR1 ligand 5-(2-oxopropylideneamino)- 6-D- ribitylaminouracil (5-OP-RU), in that they can be defined based on their MR1-dependent response to microbial infection and binding to alternate MR1 tetramers. We have generated a pipeline approach for identifying new, microbially-derived MR1 antigens, and demonstrate that MR1Ts in the lung are characterized by oligoclonal enrichments, possibly driven by these antigens. Together, these data support the specific aims of this grant which are to 1) define the repertoire of ligands presented by MR1 from M. smeg/Mtb and define the structural basis of their presentation by MR1. We focus on Mtb for its disease relevance to human health but also from our preliminary data demonstrating migration of MR1 reactive T cells to the lung during Mtb infection. Our Aim 2 is to define the T cell repertoire of MR1Ts recognizing antigens presented by MR1 from M. smeg/Mtb and define the structural basis of their recognition of the MR1- antigen complex. This is an obvious extension from preliminary data from us and others demonstrating that the MR1T population contains diversity previously unappreciated. We seek to know whether this diversity in the TCR repertoire drives antigen selectivity. Directly related to Aims 1 & 2 is our Aim 3 which will determine the biological significance of MR1-ligand/MR1T cell selectivity in human health and disease. We hypothesize that MR1T cells with a diverse TCR repertoire selectively expand at infected tissue sites in response to microbe/ligand recognition via MR1. Here our focus will be on Mtb, and we capitalize on the expertise and patient accessibility of Dr. Waltz (Capetown) to derive lung (BAL) and PBMC samples from infected and control individuals. Ultimately, the work from this project would support MR1T cell targeted vaccines and immune-therapies as a means to improve resistance to disease following exposure to Mtb.